Support with crosslinked marine collagen for tissue engineering and manufacture of biomaterials

ABSTRACT

The invention relates to a method of in vitro testing of the efficacy of a potentially active substance comprising monitoring the effect of said potentially active substance on an artificial skin, comprising a composite product forming a collagen support comprising at least one porous collagen layer covered on at least one side with a collagen membrane component selected from the group consisting of a collagen membrane prepared by compression of a collagen sponge at a pressure of at least about 50 bar and of a collagen membrane comprising a collagen film prepared by drying a collagen gel separately from the porous collagen layer, thereby providing a reliable method for finding new potentially active substances.

This application is a Divisional and Continuation in Part of U.S. patentapplication Ser. No. 09/616,282 filed on Jul. 14, 2002, and of U.S.patent application Ser. No. 09/616,526 filed on Jul. 14, 2002.

The invention relates to a support with collagen base for tissueengineering and manufacture of biomaterials and to different methodsincluding a method of in vitro testing of the efficacy of a potentiallyactive substance using such a support.

DISCUSSION OF THE PRIOR ART

For many years collagen has proved to be an irreplaceable substrate forthe production of artificial tissues containing living cells.

The biomaterials obtained are increasingly used in the field ofpharmaceutics and they appear to have a very promising future for thepreparation of injured connective tissues or for gene therapy byallowing the introduction and survival of modified cells in a livingorganism.

Furthermore, for “in vitro” tests, the cosmetic and dermopharmaceuticalindustries are increasingly using reconstructed skin, especially sinceanimal tests are used less and less in these disciplines.

It is for this reason that several research teams throughout the worldhave been endeavoring to develop collagen-based supports for theproduction of living artificial tissues such as skin, cartilage, bone,tendon or reconstructed cornea, so these novel biomaterials havenumerous fields of application.

It should be noted that the principal studies carried out in the fieldcovered by the invention are attributable mainly to the following teams:Yannas I., Collombel C., Tinois E., Boyce S., Eisenberg H., Bell E.,Kuroyanagi Y., Maruguchi T., Hanthamrongwit M., Auger F. A. and OsborneC. S.; cf. U.S. Pat. No. 5,273,900, for example.

All these researchers use of either gels, or of collagen sponges, theselast being obtained by freeze-drying (lyophilization).

It is known by document WO 99/19005 a membrane multi-layer including alayer of collagen layer with prevalence out of collagen II having asponge texture covered on at least a face and preferably on the twofaces, of at least a barrier layer having a closed texture, relativelyimpermeable.

It arises from the text that the barrier layer is consisted a naturalanimal membrane (see page 7, lines 23 to 32 and page 8, lines 10 to 30).

This barrier layer aims to prevent the penetration and thus the growthof native tissue cells because this membrane is dedicated to thereconstruction of the bone or especially of the cartilage, so that it isnecessary to use, as material prevailing of its porous layer, collagenII obtained from cartilage, preferably from hyaline cartilage of pig(see page 7, lines 14-16).

It should be noted that the cells of cartilage or the chondrocytes havea speed of multiplication or of regeneration much slower than theregeneration speed of the cells of soft tissues such as the fibroblastsand, thus, it is necessary to insulate them to enable them to grow whileavoiding being invaded by the cells with fast growth of soft tissues.This document ends in this solution by using a barrier layer tight tothe cells which protects the growth of the collagen II cells whichsupport the growth of the chondrocytes (see page 2, lines 15 to 20).

Within the framework of this further described invention, it isinitially prepared a bi-layer material of which each layer is able toallow the growth of human living cells, which constitutes an unobviousand completely unexpected solution compared to the state of the art.

The document WO 96/08277 relates to the use of a collagenic membrane asprosthesis of peritoneal regeneration, constituting a prior invention ofthe same applicant. In this document, a preferred embodiment is a mixedmembrane comprising a collagen sponge on which collagen gel was stuck,the membrane being obtained by drying the collagen gel in a nontoxicgaseous fluid, see in particular example II, pages 12 and 13 of this PCTapplication.

However, in example II, it arises that the obtained sponge is compressedduring 15 seconds under a pressure of 150 bars and that the mixedmembrane is formed by laying down a collagen gel at 1% collagen on thiscompressed sponge, this gel being then dried at ambient air.

According to the sponge compression obtained during 15 seconds under apressure of 150 bars, the obtained mixed membrane is in fact producedstarting from two primarily compact layers, which constitutes astructure different from that object of this invention. In addition, inthe case of this invention, it was discovered in an unexpected way thatthe claimed bi-layered structure was compatible with a seeding of atleast a layer with human living cells, by allowing their conservation,like their multiplication.

It should be noted that the document FR 2 679 778 constitutes anotherstill prior invention of the same applicant.

Document EP 0 686 402 still constitutes a prior document of the sameapplicant relating to an anti-adherence post-operative collagenicmembrane comprising two layers, a support containing collagen completelycovered of a layer of gelatine, the mixture being in a freeze-driedstate. It should be noted that here the critical purpose of the gelatinelayer is to carry out an effect of gluing of the membrane avoidingadherences and the gelatine has a fast resorption by dissolution at 37°C. in the presence of cells.

Document EP 0 789 074 of L'OREAL relates to an equivalent of skinsincluding the Langerhans cells.

It should be noted that in this application, the described supportitself on column 4, page 3, is an unspecified support of prior art. Itcan be formed by mixed collagen/fibroblaste lattices, a dermisbeforehand de-epidermized, an artificial membrane, a subcutaneoussubstitute containing collagen, a plastic or any other supportcompatible with cellular viability (column 4, lines 3 to 12).

This document is different from this invention insofar as it uses adermis obtained by delamination of a skin, covered with a mixture ofhuman keratinocytes previously separated according to a traditionalmethod, mixed with human melanocytes also previously separated accordingto a known method and which are then submitted to a Co-culture. Thisdocument does not foresee a compact layer as laid down within theframework of this invention and which can be seeded with human livingcells, combined in a critical way with a porous underlayer which isclearly different from a de-epidermized dermis.

Lastly, document WO 91/16010 describes equivalents of living skincomposites first of all consisting in buying in the trade a bovinecollagen sponge membrane which is inoculated with fibroblasts cells (seepage 8, third and fourth paragraphs).

After incubation, the sponge is reversed and the upper surface islaminated with nonporous collagen which can be treated with pepsin (seepage 8 last paragraph) which is in general bovine collagen. It isindicated that the purpose of the treatment with pepsin is to remove thetelopeptides (page 9, the first four lines). The pH of the collagensolution is adjusted to a neutral pH, which allows collagen toprecipitate. Collagen forms a layer of thin film on the sponge and thewhole is cultivated at 37° C. during 60 minutes (page 9, last sentenceof the first paragraph).

Then, cultivated keratinocytes are inoculated on the laminated layer anda new is culture still carried out at pH 7.2 and at 35° C. during 10days.

Within the framework of this invention, as it results from the followingdescription, the bi-layered structure is formed initially and is made upin a critical way of a collagen sponge covered of a compact layer, thiscompact layer providing unexpected technical effects as given in thefollowing description.

The main difficulties to be overcome in the production of supports forthe production of living artificial tissues are as follows: goodmechanical strength, low sensitivity to temperatures around 37° C.,biological properties favorable to cell development and metabolism, lowsusceptibility to enzymatic degradation and, finally, for certainapplications and particularly reconstructed skin, preferably thepresence of a bilayer structure in which one of the layers is as compactas possible and the other is porous.

The researches carried out hitherto have not provided collagen supportswhich satisfactorily comply with all the constraints listed above.

PURPOSES AND OBJECTS OF THE INVENTION

The object of the present invention is to solve these problems whichhave remained shelved from both the technical and industrial points ofview.

The present invention makes it possible to solve all these technicalproblems in a particularly simple, inexpensive manner applicable to theindustrial scale, particularly in cosmetics, dermopharmaceutics orpharmaceutics.

DETAILLED DESCRIPTION OF THE INVENTION

According to a first feature, the present invention provides a novelcomposite product forming a collagen support comprising at least oneporous collagen layer covered on at least one side with an essentiallycompact collagen membrane consisting either of a collagen film preparedby drying a collagen gel, preferably in air or a gaseous fluid, or of ahighly compressed collagen sponge.

According to yet another advantageous characteristic of the compositeproduct of the invention, the collagen sponge is compressed at apressure of at least about 50 bar, equivalent to about 50.10⁵ Pascals(Pa), and preferably of between 50 bar (50.10⁵ Pa) and 200 bar (200.10⁵Pa), this compression optionally taking place at a temperature ofbetween 20 and 80° C. and preferably of between 40° C. and 60° C.

According to one advantageous characteristic of this composite product,the collagen product is selected from collagen and a mixture of collagenwith a polysaccharide, particularly a glycosaminoglycan, chitosan or aderivative thereof, cellulose or a derivative thereof, dextran or aderivative thereof, an alginate or a derivative thereof, or acarrageenan.

According to another advantageous characteristic of this compositeproduct, at least one of the two layers of the latter, i.e. the porouslayer and the essentially compact membrane, comprises normal,genetically modified or malignant living cells originating particularlyfrom young or elderly subjects.

In one advantageous variant, the living cells are selected from thegroup consisting of fibroblasts, keratinocytes, melanocytes, Langerhans'cells originating from the blood, endothelial cells originating from theblood, blood cells, particularly macrophages or lymphocytes, adipocytes,sebocytes, chondrocytes, osteocytes, osteoblasts, Merkel's cellsoriginating from the blood and dendritic cells, said cells being normal,genetically modified or malignant.

According to yet another advantageous characteristic, the compositeproduct contains normal, genetically modified or malignant fibroblastsin the porous layer and normal, genetically modified or malignant livingcells on the surface of the compact membrane, said cells being selectedparticularly from keratinocytes, melanocytes, Merkel's cells originatingfrom the blood, Langerhans' cells originating from the blood, sebocytes,cells originating from the blood, nerve cells and dendritic cells.

In yet another advantageous embodiment of the invention, it can be ofparticular value to prepare either “young” reconstructed skin usingcells taken substantially exclusively from young subjects, or “aged”reconstructed skin obtained from cells taken substantially exclusivelyfrom elderly subjects. These models will make it possible to improve ourknowledge of the skin ageing process and study the influence of activeagents on this process.

In yet another advantageous embodiment of the invention, the essentiallycompact membrane is prepared prior to combination with the porous layer,preferably comprising a collagen sponge, in particular by preparing themembrane and depositing it on a collagen gel before the whole is frozenand lyophilized to give said composite product.

In yet another embodiment of the composite product according to theinvention, the collagen sponge and/or the collagen film and/or thecollagen membrane of said product comprise collagen of mammalian origin,particularly of bovine origin.

According to still another advantageous embodiment of the compositeproduct according to the present invention, the collagenic sponge and/orthe collagenic film and/or collagenic membrane, includes collagen ofmarine origin, preferably resulting from teleost fish skins, moreparticularly from fishes presenting non-pigmented skin areas, even moreparticularly from flat fishes, still better those which are fished in anindustrial way, such as for example the sole, the dab, the turbot, thebrill, the non-pigmented ventral skins of which can easily be separatedby cutting-up. The preferred fish skin as source of extraction ofcollagen usable according to the present invention is the skin of sole.The preparation of collagen starting from fish skins is in particulardescribed in the preceding document of applicant EP 0 592 586=U.S. Pat.No. 5,420,248 to which the skilled person in the art will be able torefer. It is particularly unexpected that such a collagenic spongeand/or a collagenic film and/or a collagenic membrane obtained startingfrom collagen of marine origin, preferably of teleost fishes, can bebiocompatible with human living cells used for the manufacture ofreconstructed skins and that these human living cells can not onlyremain alive but also be able to multiply.

According to another advantageous embodiment of the invention, thecollagen of mammalian origin, in particular of bovine origin, orpreferably of marine origin, in particular of teleost fish skins can beeither crosslinked chemically, or by physical crosslinking as that willbe described further within the framework of the manufacturing process.It is particularly unexpected that such a crosslinking can be usedwithin the framework of the manufacturing of a biocompatible biomaterialwith human cells which are used in the foregoing process for themanufacture of reconstructed skins.

By “biocompatible”, it is meant within the framework of this inventionthat the biomaterial is not toxic toward the human living cells and thatit allows also their growth or multiplication. It should be noted thatthe crosslinking has generally the effect to make materialnonbiocompatible, therefore toxic for the living cells and cannot thenallow their growth.

In yet another advantageous embodiment of the composite productaccording to the invention, at least one of the two layers of saidproduct is produced from a collagen gel containing a mixture of solublecollagen and insoluble collagen, for example in the form of fibers.

In the case of the composite product according to the invention, thecollagen can be type I and/or type III collagen.

According to a second feature, the present invention also covers aprocess for the manufacture of a composite product comprising at leastone porous collagen layer covered on at least one side with anessentially compact collagen membrane, wherein:

a) first of all the essentially compact collagen membrane is preparedeither by drying a first collagen gel, preferably in air or with the aidof a gaseous fluid, or by compressing a collagen sponge obtained by thefreezing-lyophilization of a collagen gel;

b) a second collagen gel is prepared separately;

c) either the essentially compact membrane is deposited on the secondcollagen gel, or the second collagen gel is poured onto the essentiallycompact membrane; and finally

d) the whole is frozen-lyophilized to give said composite product.

In one advantageous variant of this process, the collagen sponge used toprepare the compact membrane is compressed at a pressure of at least 50bar (about 50.10⁵ Pa) and preferably of between 50 bar (50.10⁵ Pa) and200 bar (200.10⁵ Pa).

The compression step advantageously takes place at a temperature ofbetween 20 and 80° C. and preferably of between 40° C. and 60° C.

In another advantageous embodiment of this process, the collagen spongeand/or the collagen film and/or the collagen membrane are prepared usingeither collagen or a mixture of collagen with a polysaccharide,particularly a glycosaminoglycan, chitosan or a derivative thereof,cellulose or a derivative thereof, dextran or a derivative thereof, analginate or a derivative thereof, or a carrageenan.

In another variant of the process, at least one of the two layers, orboth layers, are crosslinked.

In one advantageous variant, the above-mentioned crosslinking is aphysical crosslinking, particularly a thermal dehydration under vacuum,or TDH, or a chemical crosslinking, particularly withdiphenylphosphorylazide, or DPPA, with an aldehyde such asglutaraldehyde, with carbodiimide or with succinimide.

In another advantageous variant of this process, a compound which favorscell development, particularly a growth factor and especially a cytokineor a chemokine, is added during manufacture.

In another advantageous embodiment of the process according to theinvention, provision is made for a step for the introduction of normal,genetically modified or malignant living cells into at least one of thetwo layers.

In one advantageous variant, said living cells are selected from thegroup consisting of fibroblasts, keratinocytes, melanocytes, Langerhans'cells originating from the blood, endothelial cells originating from theblood, blood cells, particularly macrophages or lymphocytes,chondrocytes, osteocytes, particularly osteoblasts, Merkel's cellsoriginating from the blood, sebocytes, adipocytes, nerve cells, anddendritic cells, and any combination thereof. Said cells may be normal,genetically modified or malignant.

In one particularly advantageous embodiment of the invention, theprocess comprises introducing fibroblasts into the porous layer.

In a more preferred embodiment of the invention, the process comprisesdepositing living cells on the surface of the compact membrane, saidcells being selected particularly from keratinocytes, melanocytes,Merkel's cells originating from the blood, Langerhans' cells originatingfrom the blood, sebocytes, cells originating from the blood, nerve cellsand dendritic cells.

In one variant of the process of the invention, the living cells areprovided either by the sequential culture or by the concomitant cultureof the different types of cells, these cells originating from culture orbiopsy.

According to a third feature, the present invention also covers the useof the composite product forming a collagen support as defined above, oras obtained by the process defined above, or as resulting from thefollowing description relating especially to the Examples, for whichevery characteristic which appears to be novel compared with any stateof the art is claimed as such in its function and in its generality, forthe manufacture of artificial skin intended especially for performing invitro tests on the efficacy of a potentially active substance or forreconstructing damaged areas of skin in vivo.

According to one advantageous characteristic, the artificial skin can beobtained either substantially exclusively from young cells orsubstantially exclusively from aged cells, in particular for studyingthe tissue ageing process, especially the skin ageing process, andoptionally for testing the efficacy of active principles on thisprocess.

Thus it is seen that the invention provides a solution to theabove-mentioned technical problems.

To obtain the strongest collagen materials, the inventors carried outmore particularly the process described in U.S. Pat. No. 5,333,092granted on 19 Jul. 1994. This technique affords a mixture of soluble andinsoluble type I and type III native collagens which are very strongfrom the mechanical point of view and very resistant to enzymaticdigestion. These last two characteristics may optionally be improved byany crosslinking technique or by the addition of substances whichinteract strongly with collagen and do not exhibit toxicity towards thecells. Furthermore, this collagen production process makes it possiblevirtually to eliminate the risk of biological contamination due tobacteria, viruses or prions.

For the case of supports intended for obtaining reconstructed skin, theinventors came to the idea of preparing bilayer materials by producingfirstly the more compact layer and then the porous sponge. Thismethodology has the advantage of resulting in a much more compactsurface layer than all those described hitherto. In particular, spongescompacted by high compression, or films, can therefore be fixed toporous matrices.

The use of the supports described above for tissue engineeringapplications involves the inoculation of living or genetically modifiedcells, it being possible for the cells to develop either inside thecollagen support or on its surface.

The reconstructed living tissues obtained in this way can be used innumerous cosmetic, dermopharmaceutical or pharmaceutical applicationsas:

-   -   “in vitro” models for simulating the effects of ingredients on        cell metabolisms for the purpose of evaluating the efficacy and        toxicity of raw materials or more complex formulations;    -   reconstructed tissues capable of overcoming the deficiencies of        damaged tissues: skin, cartilage, bone, tendon, comea; or    -   living implants containing modified cells capable of overcoming        certain deficiencies of the organism, particularly in the field        of gene therapy.

Other objects, characteristics and advantages of the invention willbecome clearly apparent from the following explanatory descriptionreferring to various Examples of the invention, which are given simplyby way of illustration and cannot therefore in any way limit the scopeof the invention. As indicated previously, any characteristic in theExamples which appears to be novel compared with any state of the art isclaimed in its function and in its generality, independently of thecontext of the Example. Moreover, Examples 6 to 13 constitute currentlypreferred embodiments of the composite products according to theinvention which form a collagen support. Example 14 refers tocomparative tests demonstrating the value of the composite productsaccording to the invention as collagen supports for the manufacture ofartificial skin intended especially for performing in vitro tests on theefficacy of a potentially active substance or for reconstructing damagedareas of skin in vivo.

IN THE ATTACHED FIGURES

FIG. 1 shows a sectional view, after marking by conventionalhistological staining, of a composite product according to the presentinvention which has been produced from a porous lower layer of bovinecollagen covered on the top side with an essentially compact uppercollagen membrane consisting of a collagen film prepared by drying acollagen gel in air under the conditions of Example 6;

FIG. 2 shows a similar section obtained with a simple porous collagenlayer which has been prepared with the same bovine collagen gel, exceptthat it has not been covered, i.e. under the conditions of Example 1,showing the presence of deep inclusions of keratinocytes not limited tothe surface;

FIG. 3 shows the quantity of laminins present in the reconstructed skinincubation media, respectively for young reconstructed skin obtainedfrom cells taken from donors of 25 to 35 years of age, and for mature oraged reconstructed skin obtained from cells taken from donors of morethan 55 years of age, in order to show the influence of the donor's age,the results being expressed in the form of “bars” and the quantity oflaminins produced being expressed on the ordinate as a percentage of thecontrol (YRS=young reconstructed skin);

FIG. 4 shows the inductive effect of a fermented malt extractcommercially available under the trade mark BASALINE®, COLETICA, France,on the production of laminins in mature reconstructed skin, the quantityof laminins produced again being expressed as a percentage of thecontrol; and

FIG. 5 shows the compensating effect of the same fermented malt extract,or BASALINE®, the quantity of laminins produced being expressed on theordinate as a percentage of the control.

FIG. 6 shows the proliferation of the normal human fibroblasts in dermisequivalent, with the time expressed in day and in abscissa and theoptical density×1000 in ordinates with units increasing by 100; thecurve with the rhombuses is that which is obtained by using as support aaquatic collagen porous collagen layer, here of fishes, and the curvewith squares is obtained with bovine collagen; and

FIG. 7 shows a similar curve of proliferation of fibroblasts in dermisequivalent with the time expressed in day in abscissa and thefluorescence expressed in international unit in ordinates, starting from15,000 with units increasing by 10,000; the curve with the fullrhombuses represents the fluorescence obtained within the framework oftest 1; the curve with the square that obtained with test 2; the curvewith the empty triangles being obtained with test 3 and finally thecurve with the crosses being that obtained with test 4.

EXAMPLE 1 OF THE INVENTION Preparation of a Porous Collagen Layer ofNative Collagen by the Technique of U.S. Pat. No. 5,331,092

A—Preparation of the Native Collagen

A gel is prepared from calf skins which have previously been washed (2hours) and then depilated with a lime/sulfide mixture (lime: 3.5%,sodium sulfide: 2.5%) at a rate of 400 g of skin (solids content: about30%) to 250 ml of water. This bath lasts for 30 minutes with rotation at4 rpm.

The total depilation time is 36 hours.

The skins are then unlimed in a bath containing ammonium chloride (3%)and sodium metabisulfite (0.5%) at a rate of 400 g of skin to 50 ml ofbath.

The total duration of this bath is 2 hours thirty minutes.

The salts are removed by two successive washes with water (15 minutesper wash) at a rate of 200 ml of water to 100 g of tissue.

The skins are subsequently ground and then washed by agitation for 1hour with phosphate buffer of pH 7.8 (0.78 g/l of potassiumdihydrogenphosphate and 21.7 g/l of disodium monohydrogenphosphate) at arate of 5 l buffer/kg ground material. The phosphate is then removed bytwo successive washes with softened water and then by continuouscentrifugation at 4000 rpm (Rousselet centrifuge) at a rate of 5 l ofwater per kg of ground material.

The ground material is then acidified with 10% acetic acid solution, theamount of acetic acid being 5% based on the collagen; the final molarityis about 0.08 M.

The ground material is then malaxated for one hour to give a paste.

The gel is obtained by continuously passing the paste through a UTL T/-6ultrasonic treatment apparatus. This gel has a concentration of between0.7 and 2% of collagen, the proportion of acid-soluble collagen varyingfrom 10 to 20% based on the insoluble collagen.

B—Preparation of the Porous Collagen Layer with the Collagen GelObtained as Indicated Above

20 g/cm² of collagen gel (solids content=0.75%) are placed in alyophilization tray and lyophilized by freezing at −30° C. and thenheating at +32° C. The total lyophilization time is 16 hours under apressure of 400 microbar.

The lyophilizate is crosslinked by a physical method (TDH), thelyophilizate being placed for 10 hours in an oven at 110° C. and 400microbar of pressure.

EXAMPLE 2 OF THE INVENTION Preparation of a Porous Collagen LayerCrosslinked with Diphenylphosphorylazide (DPPA) by the TechniqueDescribed in European Patent No. 466 829 of 24 Jul. 1996

The collagen lyophilizate is incubated for 24 h in a solution containing5 to 250 μl DPPA/g collagen in 100 ml of dimethylformamide (DMF). Thecollagen is then rinsed in 100 ml of DMF to remove the DPPA. The DMF isthen removed by rinsing in 100 ml of a borate buffer solution of pH 8.9(0.04 M sodium tetraborate, 0.04 M boric acid).

The collagen is finally incubated overnight in the same borate buffer,the borate buffer then being removed by continuous rinsing with softenedwater for 6 h.

EXAMPLE 3 OF THE INVENTION Preparation of a Porous Collagen LayerCrosslinked with Carbodiimide and N-hydroxysuccinimide

The collagen is crosslinked with EDC(ethyldlmethylaminopropylcarbodilmide) at a concentration of 0.23 to0.69 g/g collagen and with NHS (N-hydroxysuccinimide) at a concentrationof 0 to 0.42 g/g collagen.

After rinsing with softened water, the collagen is lyophilized again.

EXAMPLE 4 OF THE INVENTION Preparation of a Porous Collagen LayerCrosslinked with Glutaraldehyde

The collagen is crosslinked for 24 to 96 h in a solution containing 0.6to 1% of GTA at 20° C.

After rinsing with softened water, the collagen is lyophilized again.

EXAMPLE 5 OF THE INVENTION Porous Collagen Layer Prepared with theNative Collagen of Example 1 in Association with Chitosan and aGlycosaminoglycan as Described in European Patent No. 296078 of 29 May1991

A solution of 2.5 g of chitosan in 356 ml of water and 1.9 ml of aceticacid, and then a solution containing 1 g of chondroitin 4-sulfate in 400ml of softened water, are added to 600 g of 1.5% collagen gel. Themixture, which has a pH of about 4.0, is subsequently agitated and thenlyophilized.

The sponge obtained is crosslinked by TDH.

EXAMPLE 6 OF THE INVENTION Porous Collagen Layer Described in Example 1,Covered with a Collagen Film

A—Preparation of the Film

Collagen gel with a solids content of between 0.3 and 0.8% is dried inan oven at 30° C. or under a hood at a rate of 0.5 g gel/cm² tray. 10 to40% of glycerol can be added to the collagen gel.

The collagen dried under these conditions forms a transparent film.

B—Association of the Film with the Porous Collagen Layer Described Above

0.5 g/cm² of collagen gel of example 1 with a solids content of 0.75% isplaced in a lyophilization tray, the collagen film is then deposited onthis gel and the whole is lyophilized.

The lyophilizate obtained is crosslinked by TDH.

EXAMPLE 7 OF THE INVENTION Porous Collagen Layer Prepared with anAcid-Soluble Collagen Gel and Covered with a Collagen Film

The process is that indicated in Example 6, the only difference being inthe nature of the gel poured onto the film, which consists ofacid-soluble collagen prepared by a technique well known to thoseskilled in the art.

EXAMPLE 8 OF THE INVENTION Porous Collagen Layer Prepared with anAtelocollagen Gel and Covered with a Collagen Film

The process is that indicated in Example 6, the only difference being inthe nature of the gel poured onto the film, which consists ofatelocollagen, i.e. telopeptide-free collagen prepared by a techniquewell known to those skilled in the art.

EXAMPLE 9 OF THE INVENTION Porous Matrix Collagen Layer Consisting ofCollagen Associated with Chitosan and a Glycosaminoglycan and Coveredwith a Collagen Film

The process is that indicated in Example 6 except that in this case thegel poured onto the collagen film consists of collagen, chitosan and aglycosaminoglycan. The preparation of this gel is described in Example5.

EXAMPLE 10 OF THE INVENTION All the Porous Matrices Described Above,Covered with a Collagen Film, can be Crosslinked by the TechniquesDescribed in Examples 2, 3 and 4 EXAMPLE 11 OF THE INVENTION PorousMatrix of Collagen Only, Described in Example 1, Covered with aCompressed Collagen Sponge

A—Preparation of the Compressed Sponge

Collagen gel prepared as in Example 1, with a solids content of between0.3 and 1.5%, is lyophilized to give a sponge weighing between 0.5 and 2g/cm².

The lyophilizate is compressed for 5 to 60 seconds at a temperature ofbetween 20 and 60° C. and a pressure of between 50 and 200 bar (50 to200.10⁵ Pa).

B—Association of the Compressed Sponge with the Porous Matrix

The collagen gel described in Example 1 is deposited in a lyophilizationtray at a rate of 0.5 g per cm². The compressed sponge is then depositedon this gel and the whole is lyophilized to give a porous collagensponge covered with a compressed collagen sponge. The whole iscrosslinked by TDH as described in Example 1.

EXAMPLE 12 OF THE INVENTION Porous Matrix Consisting of Collagen,Chitosan and Glycosaminoglycan, as Described in Example 5, Covered withCompressed Sponge

The collagen, chitosan and glycosaminoglycan gel prepared by the processof Example 5 is deposited in a lyophilization tray at a rate of 0.5 gper cm², the compressed sponge is then deposited on this gel and thewhole is lyophilized. The lyophilizate is then crosslinked by TDH asdescribed in Example 1.

EXAMPLE 13 OF THE INVENTION

All the porous matrices described above, covered with a compressedcollagen sponge, can be crosslinked by the techniques described inExamples 2, 3 and 4.

EXAMPLE 14 OF THE INVENTION

Reconstructed skin prepared either with the aid of the DPPA-crosslinkedporous matrix described in Example 2, or with the aid of theDPPA-crosslinked porous matrix of Example 2 covered with a compressedcollagen sponge, the whole being crosslinked with DPPA, according toExample 13, in order to allow a comparison to be made between acomposite product comprising a porous collagen layer covered with anessentially compact membrane according to the invention and a productcomprising a porous collagen layer only, with no covering

Preparation of Reconstructed Skin

a) Culture of Normal Human Fibroblasts

Normal human fibroblasts taken arbitrarily from elderly or youngsubjects are used; they are recovered and developed in a mannerconventional to those skilled in the art for recovery between the sixthand tenth passages.

Inoculation is carried out at a rate of 250,000 cells per cm² of porousmatrix, the latter being either the comparison product comprising onlythe DPPA-crosslinked porous matrix of Example 2, or the compositeproduct according to the invention comprising the DPPA-crosslinkedporous matrix of Example 2 covered with a compressed collagen sponge,the whole being crosslinked with DPPA, according to Example 13.

The culture medium is composed of DMEM/HAM F12 50/50 (v/v) supplementedwith 10% by weight of fetal calf serum, 100 IU/ml of penicillin, 25μg/ml of gentamycin, 1 μg/ml of amphotericin B and 50 μg/ml of vitaminC.

Culture is carried out for three weeks, the medium being changed threetimes a week.

b) Culture of Normal Human Keratinocytes

Normal human keratinocytes obtained arbitrarily from young or elderlysubjects are then cultured; they are recovered and cultivated by theculture techniques well known to those skilled in the art for recoverybetween the first and third passages.

Inoculation is carried out at a rate of 250,000 cells per cm² ofsurface, which is either the surface of the DPPA-crosslinked porousmatrix of Example 2, or the surface of the composite product accordingto the invention comprising the DPPA-crosslinked porous matrix ofExample 2 covered with a compressed collagen sponge, the whole beingcrosslinked with DPPA, according to Example 13, in which case thekeratinocytes are inoculated onto the surface of the essentially compactcollagen membrane.

The culture of these products, comprising an inoculation of bothfibroblasts and keratinocytes, takes place in a Green's medium composedof:

DMEM supplemented with:

30% of HAM F12,

10% of fetal calf serum,

100 IU/ml of penicillin,

100 μg/ml of streptomycin,

1 μg/ml of amphotericin B,

2 μmol/ml of L-glutamine,

10 ng/ml of EGF (Epidermal Growth Factor),

0.12 IU/ml of insulin commercially available under the trade markUMULINE®,

400 ng/ml of hydrocortisone,

10⁻¹² mol/ml of cholera toxin,

5 μg/ml of transferrin,

2.10⁻⁹ M triiodothyronine,

1.8.10⁻⁷ mol/ml of adenine,

50 μg/ml of vitamin C.

This culture is carried out for one week, the media being changed everyday.

c) Culture of the Composite Product According to the Invention and theComparative Non-Covered Porous Layer

After the culture of step b) has been carried out for one week with themedia being changed every day, the surface layer containing thekeratinocytes is caused to emerge at the air-liquid interface, while thelayer containing the fibroblasts remains immersed, and culture is thencarried out for three weeks in an emersion medium composed of:

DMEM supplemented with:

10% of fetal calf serum,

100 IU/ml of penicillin,

100 μg/ml of streptomycin,

1 μg/ml of amphotericin B,

2 μmol/ml of L-glutamine,

10 ng/ml of EGF,

0.12 IU/ml of insulin of trade mark UMULINE®,

400 ng/ml of hydrocortisone,

50 μg/ml of vitamin C.

The total culture time of 7 weeks resulting from steps a) to c) gives areconstructed skin composed of a reconstructed dermis, the fibroblastshaving colonized the three-dimensional collagen matrix, said dermisbeing covered with a multilayer epidermis.

The dermo-epidermal interface shows the presence of a basal membrane inwhich it is possible to identify the presence of laminin-1, laminin-5,type IV collagen and type VII collagen by immunolabeling.

Thus, after three weeks of preparation of the dermis equivalent,covering of the porous matrices with an essentially compact layer togive a composite product according to the invention affords a greaterquantity of fibroblasts on the surface of the collagen matrices beforeepidermization.

In the case of a porous matrix only, i.e. with no covering, if thesurface layer of fibroblasts is not completely contiguous, keratinocytescan infiltrate the underlying dermis equivalent and form islets ofkeratinocytes, which are totally abnormal features.

Thus it is seen that the invention, which uses more compact layers thanthose previously available for use in the prior art, provides bettersecurity against the penetration of keratinocytes.

It is pointed out that the abbreviation DMEM in the description denotesDulbecco Modified Eagle's Medium.

EXAMPLE 15 OF THE INVENTION Study Comparing Skin Reconstructed fromCells of Young Donors and Skin Reconstructed from Cells of ElderlyDonors with the Composite Products According to the Present Invention inOrder to Measure the Efficacy of Active Principles on the Production ofLaminins

The procedure in this Example is essentially as described in Example 14as regards the cultures, using the same composite product according tothe present invention comprising a DPPA-crosslinked porous collagenlayer or matrix described in Example 2, covered with a compressedcollagen sponge, the whole being crosslinked with DPPA, according toExample 13. The procedure is as follows:

1) Preparation of the Reconstructed Skin

Young reconstructed skin was prepared by the procedure described inExample 14 except that the fibroblasts and keratinocytes originatedrespectively from young donors, i.e. those of between 25 and 35 years ofage. Also, aged or mature reconstructed skin was obtained by usingfibroblasts or keratinocytes originating from elderly donors of morethan 55 years of age.

a) Materials and Method

As indicated in Example 14, step a, porous matrices of the compositeproduct of the invention were first inoculated with normal human dermalfibroblasts originating either from pools of young cells or from poolsof mature or aged cells, and culture is carried out for 21 days underthe conditions described in Example 14, step a.

b) After the above-mentioned 21 days of culture, epidermal layersprepared separately from keratinocytes originating either from pools ofyoung cells or from pools of mature cells are inoculated onto thesurface of the essentially compact collagen membrane of the compositeproduct.

Culture is carried out for 14 days under the conditions described inExample 14b.

2) Quantification of the Laminins

After the 14 days of culture of the fibroblast-keratinocyte composite,the laminins contained in the incubation media of the resulting young ormature reconstructed skin are quantified with the aid of a commerciallyavailable ELISA kit (Takara, Japan).

These results are reported in FIG. 3.

FIG. 3 shows that the mature reconstructed skin contains about half asmuch laminins as the young reconstructed skin (YRS) used as 100%control.

3) Measurement of the Inductive Effect of an Active Principle, Such as aFermented Malt Extract Marketed by COLETICA Under the Trade MarkBASALINE®, on the Production of Laminins in Young and MatureReconstructed Skin

In this comparative test, the procedure is as described above except inregard to the 14 days of culture with keratinocytes; the young or maturereconstituted skin is maintained in culture for 14 days either in theabsence (control) or in the presence of 0.5% by weight of fermented maltextracts commercially available under the trade mark BASALINE®,COLETICA, France.

At the end of the incubation period, as in the above Example, thelaminins contained in the incubation media were quantified by ELISA.

The results are reported in FIG. 4.

The 100% control consists of the proportion of laminins in AgedReconstructed Skin, or ARS.

FIG. 4 shows that the active principle extracted from fermented malt, orBASALINE®, was capable of stimulating laminin production in maturereconstructed skin. Under the same conditions, the active principleextracted from fermented malt, or BASALINE®, does not significantlymodify laminin production in young reconstructed skin, as indicated inFIG. 5.

Thus it is seen that the active principle extracted from fermented malt,or BASALINE®, increases laminin production in mature reconstructed skinby 65%.

By the same token, this active principle does not affect thephysiological processes involved in the regulation of laminin productionin young reconstructed skin.

These experiments made it possible to evaluate the magnitude of thecompensating effect of the fermented malt extract, or BASALINE®, definedas the capacity of this active principle to reduce the relativedifference observed between laminin production in young reconstitutedskin and that in mature reconstituted skin.

FIG. 5 shows that the difference between laminin production in youngreconstituted skin and that in mature reconstituted skin can be reducedby 65% when the active principle is used at 0.5%.

EXAMPLE 16 Preparation of a Porous Matrix of Aquatic Native Collagen

The collagen is obtained by the technique of U.S. Pat. No. 5,331,092granted on 19 Jul. 1994.

A—Preparation of the Aquatic Native Collagen

A collagen gel is prepared from ventral sole skin which is ground andthen washed with a phosphate buffer of pH 7.8 having the followingcomposition: 0.78 g/l of potassium dihydrogenphosphate and 21.7 g/l ofdisodium monohydrogenphosphate. The washing is carried out withagitation for one hour at a rate of 5 l of buffer per kg of groundmaterial. The phosphate is then removed by means of two successivewashes with softened water, followed by continuous centrifugation at4000 rpm (Rousselet centrifuge), at a rate of 5 l of water per kg ofground material.

The ground material is then acidified with 0.25 M acetic acid solutionat a rate of 1 kg of ground material to 10 l of solution. The gel isthen centrifuged at 4000 rpm for 5 min.

The gel to be used consists of the supernatant obtained, which has acollagen concentration of between 0.5 and 2%.

B—Preparation of the Porous Matrix from the Collagen Gel Obtained Above

This gel is poured into a lyophilization tray at a rate of 20 g/cm². Itis then lyophilized after freezing at −30° C. and heating at +32° C.

The total lyophilization time is 16 hours under a pressure of 400microbar. The matrix obtained is then crosslinked by thermal dehydrationCTDH), which consists in heating in an oven at 110° C. under a vacuum of400 microbar for 16 hours.

EXAMPLE 17 Preparation of a Porous Matrix Crosslinked withDiphenylphosphorylazide (DPPA) by the Technique Described in EuropeanPatent No. 466 829 of 24 Jul. 1996

The collagen matrix of Example 1 is incubated for 24 h in a solutioncontaining 5 to 250 μl DPPA/g collagen in 100 ml of dimethylformamide(DMF). The collagen is then rinsed in 100 ml of DMF to remove the DPPA.The DMF is then removed by rinsing in 100 ml of a borate buffer solutionof pH 8.9 (0.04 M sodium tetraborate, 0.04 M boric acid).

The collagen is finally incubated overnight in the same borate buffer,the borate buffer then being removed by continuous rinsing with softenedwater for 6 h.

EXAMPLE 18 Preparation of a Porous Matrix Crosslinked with Carbodiimideand N-hydroxysuccinimide

The aquatic collagen matrix of Example 1 is crosslinked with EDC(ethyldimethylaminopropylcarbodiimide) at a Concentration of 0.23 to0.69 g/g Collagen and with NHS (N-hydroxysuccinimide) at a Concentrationof 0.42 g/g Collagen.

After rinsing with softened water, the collagen is lyophilized again.

EXAMPLE 19 Preparation of a Porous Matrix Crosslinked withGlutaraldehyde

The porous matrix of aquatic collagen of Example 1 is crosslinked for 24to 96 h in a solution containing 0.6 to 1% of GTA at 20° C. Afterrinsing with softened water, the collagen is lyophilized again.

EXAMPLE 20 Porous Matrix Prepared with the Aquatic Native Collagen ofExample 16 in Association with Chitosan and a Glycosaminoglycan asDescribed in European Patent No. 296078 of 29 May 1991

A solution of 2.5 g of chitosan in 356 ml of water and 1.9 ml of aceticacid, and then a solution containing 1 g of chondroitin 4-sulfate in 400ml of softened water, are added to 600 g of 1.5% collagen gel. Themixture, which has a pH of about 4.0, is subsequently agitated and thenlyophilized.

The sponge obtained is crosslinked by TDH.

EXAMPLE 21 Porous Matrix Described in Example 16, Covered with aCollagen Film

A—Preparation of the Film

The collagen gel, which has a solids content of between 0.3 and 0.8%, isdried in an oven at 30° C. or under a hood at a rate of 0.5 g gel/cm²tray.

10 to 40% of glycerol can be added to the collagen gel.

The collagen dried under these conditions forms a transparent film.

B—Association of the Film with the Porous Matrix Described above

The aquatic native collagen gel with a solids content of 0.5% to 2% isdeposited in a lyophilization tray at a rate of 0.5 g per cm², thecollagen film is then deposited on this gel and the whole islyophilized.

The lyophilizate obtained is crosslinked by TDH.

EXAMPLE 22 Porous Matrix of Collagen Only, Described in Example 16,Covered with a Compressed Collagen Sponge

A—Preparation of the Compressed Sponge

The collagen gel prepared as in Example 1, with a solids content ofbetween 0.3 and 1.5%, is lyophilized to give a sponge weighing between0.5 and 2 g/cm².

The lyophilizate is compressed for 5 to 60 seconds at a temperature ofbetween 20 and 60° C. and a pressure of between 50 and 200 bar (50 to200.10⁵ Pa).

B—Association of the Compressed Sponge with the Porous Matrix

The collagen gel described in Example 1 is deposited in a lyophilizationtray at a rate of 0.5 g per cm². The compressed sponge is then depositedon this gel and the whole is lyophilized to give a porous collagensponge covered with a compressed collagen sponge. The whole iscrosslinked by TDH as described in Example 1.

EXAMPLE 23 Porous Matrix Consisting of Collagen, Chitosan andGlycosaminoglycan, as Described in Example 20, Covered with CompressedSponge

The collagen, chitosan and glycosaminoglycan gel prepared by the processof Example 20 is deposited in a lyophilization tray at a rate of 0.5 gper cm², the compressed sponge is then deposited on this gel and thewhole is lyophilized. The lyophilizate is then crosslinked by TDH asdescribed in Example 16.

EXAMPLE 24

All the porous matrices described above, covered with a compressedcollagen sponge, can be crosslinked by the techniques described inExamples 17, 18 and 19.

EXAMPLES 25 TO 27 Tests for Comparing the Cell Metabolism of Bovine andAquatic Collagen Matrices EXAMPLE 25 Test for Cell Viability ofFibroblasts

I—Preparation of the Dermis Equivalents

For this comparative test, a DPPA-crosslinked aquatic porous matrixaccording to Example 17 is prepared first.

By way of comparison, a comparative porous matrix called a bovinematrix, also crosslinked with DPPA, is prepared with collagen of bovineorigin under the same conditions as those of Example 17.

Normal human fibroblasts, taken from a young donor pool used at the 7thpassage, are inoculated into each of the aquatic and bovine matrices ata rate of 250,000 cells per cm² in the case of the proliferation andprotein synthesis study, and at a rate of 300,000 cells per cm² in thecase of the aquatic and bovine matrices intended for the histologicalstudies.

These aquatic and bovine matrices are cultured in a medium composed ofDMEM/HAM F12 in a ratio of 50/50 (v/v) supplemented with 10% of fetalcalf serum, 100 IU/ml of penicillin, 25 μg/ml of gentamycin, 1 μg/ml ofamphotericin B and 50 μg/ml of vitamin C.

This culture is carried out for 1 month, the culture medium beingchanged 3 times a week.

II—Analyses Performed

1) Measurement of Cell Viability by Reaction with MTT

1% by weight of MTT (i.e.3-(4-(dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) is addedto the culture medium.

Incubation is carried out for 2.5 hours at 37° C.

After this incubation period, the conversion product (formazan blue) issolubilized in DMSO and its optical density is read at 550 nm.

The optical density obtained is proportional to the activity of thesuccinate dehydrogenases, which are capable of converting the brightyellow tetrazolium salt, MTT, to blue crystals of formazan.

The cell viability was measured after 1, 5, 7 and 22 days and one monthof culture.

To determine the mean values, 6 samples were prepared for each matrix.TABLE I RESULTS Mean Mean Aquatic standard Bovine standard Days matrixdeviation matrix deviation 1 487 24 403 40 5 604 19 393 59 7 520 56 39864 22 608 30 680 40

These results are also used for the curves in the attached FIG. 6.

It will be noted that the curve with the diamonds is that obtained withthe aquatic matrix and the curve with the squares is that obtained withthe bovine matrix.

The results show, totally surprisingly, that the aquatic matrixconstitutes a support which allows not only the survival of normal humanfibroblasts but also the proliferation of these normal humanfibroblasts, while at the same time even constituting a much betterculture support during the first three weeks.

It can therefore be concluded from these tests that, surprisingly,aquatic collagen is particularly suitable for the production of a tissueengineering support, in particular for applications in vitro and even,above all, in vivo for forming biomaterials containing living cells,particularly and preferably those of human beings.

2) Measurement of Protein Synthesis

The synthesis of proteins secreted over 3 days in a culture medium freeof fetal calf serum was evaluated after one month of maturation of thedermis equivalents as obtained after one month of culture under theconditions reported above in the preparation of the dermis equivalents.

The assay is performed by the microBCA method of Pierce.

The cell density was evaluated in parallel by an MTT test under theconditions described above.

The relative protein content corresponds to the protein content per unitof cell density expressed as the optical density, or OD, so that thecell concentration in question is equivalent. The results obtained areshown in Table II below: TABLE II RESULTS OF PROTEIN SYNTHESIS Aquaticmatrix Bovine matrix Collagen of the support Mean * Mean * Cell density(OD) 2.12 0.09 1.91 0.13 Proteins (μg/ml) 494 48 499 32 Relative protein233 18 262 23 content*: Mean standard deviation

As in Table I, the mean is based on 6 samples.

3) Histology

The dermis equivalents obtained after culture of the aquatic and bovinecollagen matrices for 21 days are fixed in 2% paraformaldehyde solutionand then post-fixed in osmium tetroxide solution, dehydrated, includedin Epon, sectioned and observed by transmission electron microscopy(Jeol 1200) at CMEAGB (Lyon, France).

Conclusions

These results indicate a very good colonization of the three-dimensionalmatrices, whether they be aquatic or bovine. After three weeks ofculture, the cell density is equivalent in both types of matrices.However, the aquatic matrix seems to allow a better cell adhesion at thebeginning of the experiment, as indicated by the proliferation study inthe first week of culture, and hence a better colonization for shortculture times.

As far as the protein syntheses are concerned, the fibroblast synthesiscapacities (relative protein contents) are also equivalent after onemonth of culture.

These results indicate that the aquatic collagen matrices developed madeit possible to prepare dermis equivalents of good quality, the resultsobtained with these matrices being comparable to those obtained withbovine collagen matrices.

In transmission electron microscopy, fibroblasts could be observed inthe matrices of bovine and aquatic origin. In both types of matrix, thepresence of a copious neosynthesized extracellular matrix is noted. Theneosynthesized extracellular matrix can be differentiated by virtue ofthe periodic striation of the fibers of deposited collagen, comparedwith the collagen clusters forming the three-dimensional matrix of theinitial sponge.

EXAMPLE 26 Influence of the Different Types of Crosslinking of theAquatic Collagen Matrices on the Cell Viability

The following tests are carried out in order to study the influence ofthe different types of crosslinking of the aquatic collagen matrices onthe cell viability:

I) Preparation of the Dermis Equivalents

a) Support or Matrix Used

Various collagen supports or matrices are prepared using differentproportions of collagen in the collagen gel for producing the porouslayer or matrix, and optionally using a different crosslinking agent, asfollows:

1) Test 1

For this test, a porous matrix in the form of a porous sponge isproduced from an aquatic collagen gel prepared from 1.3% by weight ofaquatic collagen, which is frozen at −80° C., subjected to standardlyophilization according to Example 17 and then crosslinked with DPPA ina proportion of 250 μl per g of sponge in the dry state.

2) Test 2

For this test, a porous support in the form of an aquatic sponge isprepared from an aquatic collagen gel comprising 0.7% by weight ofaquatic collagen, which is frozen at −80° C. and then subjected tostandard lyophilization and crosslinked with DPPA in a proportion of 250μl per g of dry sponge as in test 1.

3)Test 3

For this test, the procedure is as in Test 1 except that thecrosslinking is carried out with EDC, according to Example 2, in aproportion of 0.46 g per g of dry sponge.

4) Test 4

A porous support is prepared which comprises a sponge of aquaticcollagen obtained from an aquatic collagen gel comprising 1.1% by weightof aquatic collagen, which is frozen at −80° C. and then subjected tostandard lyophilization and crosslinked with DPPA in a proportion of 250μl per g of dry sponge as in Test 2, the difference being in theproportion of 1.1% by weight of aquatic collagen.

In all these tests, the aquatic collagen originates from ventral soleskin as in Example 17.

b) Culture of Fibroblasts on These Matrices

Normal human fibroblasts are used as in Example 25, but these are takenat the 8th passage.

Inoculation is carried out at a rate of 275,000 cells per cm².

The culture medium is composed of DMEM/HAM F12 50/50 (v/v) supplementedwith 10% by weight of fetal calf serum, 100 IU/ml of penicillin, 25μg/ml of gentamycin, 1 μg/ml of amphotericin B and 50 μg/ml of vitaminC.

Culture is carried out for 1 month, the medium being changed 3 times aweek.

4 matrices are used for each test so as to take a mean for each type oftest and measure the mean standard deviation.

II) Analyses Performed

Measurement of the Cell Viability by Reaction with Alamar Blue (RedoxMarker)

Alamar blue is added at a rate of 2% by weight of the culture mediumused, at the moment when it is desired to measure the cell viability ona sample taken from the culture medium.

After incubation for 2 h 20 min at 37° C., the fluorescence is read onthe basis of an excitation at 530 nm and an emission at 590 nm.

The intensity of the fluorescence obtained is proportional to themetabolic activity of the cells.

The cell viability is measured on 10 samples after 1, 4, 6, 11 and 17days of culture.

The results are expressed in Table III below.

The results are indicated in international units of fluorescence as afunction of time. TABLE III CELL VIABILITY (IU of fluorescence) TimeTEST 1 TEST 2 TEST 3 TEST 4 (days) Mean SD* Mean SD* Mean SD* Mean SD* 121,734 1184 30,535 1888 25,528 6820 28,461 3805 4 31,611 920 35,623 354436,404 3570 45,126 2930 6 43,144 2500 35,244 2095 37,819 4170 41,2543396 11 42,808 1481 38,532 2537 42,442 3112 44,508 2329 17 45,484 242645,094 1470 43,963 8285 43,939 4521 Order 1 2 3 4*Standard deviation

The results in Table 3 are also used in the attached FIG. 7.

They show the curves of fibroblast proliferation in dermis equivalents.

The curve with solid diamonds corresponds to Test 1, the curve withsolid squares corresponds to Test 2, the curve with trianglescorresponds to Test 3 and the curve with crosses corresponds to Test 4.

The time is expressed in days on the abscissa and the fluorescence isexpressed in IU with a scale starting at 15,000 and increasing to 55,000in units of 10,000.

The results allow the following conclusions to be drawn.

Conclusions

The results indicate that the different matrices prepared can permit agood growth of fibroblasts after 17 days of culture. Irrespective of thepreparation of the aquatic collagen matrices, the fibroblasts adherewell to their three-dimensional support and divide very rapidly tocolonize the matrix.

The proliferation profile varies very slightly from one type of matrixto the other, but the fibroblast density is comparable after 17 days ofculture, irrespective of the preparative process.

The different types of crosslinking employed, carried out either withDPPA or with EDC, do not seem to influence the cell renewal. Afterpractically 3 weeks of culture, the stability of the matrices isexcellent, there being little digestion and little contraction.

EXAMPLE 27 Test Demonstrating the Advantages of Aquatic Collagen for theIdentification and Assay of Neosynthesized Human Collagen

This test is similar to that of Example 25 except that histology iscarried out with immunolabeling.

The test is performed as follows:

1) Preparation of the Dermis Equivalents

These are the dermis equivalents of Example 25, culture being carriedout under the conditions of Example 25.

This culture is therefore carried out for three weeks with the mediumbeing changed three times a week, the normal human fibroblasts havingbeen inoculated at a rate of 300,000 cells per cm², as indicated inExample 25.

2) Histology

a) Conventional Histology

Fixing is effected with paraformaldehyde at a concentration of 4% byweight, after which the material is dehydrated and included in paraffin.

This is followed by the preparation of 7 μm sections and MalloryHaidenhain staining after removal of the paraffin and rehydration.

b) Immunolabeling

Fixing is again effected with 4% by weight of paraformaldehyde, thematerial is included in Tissue Tek OCT compound, i.e. an inclusionliquid supplied by Miles, Elkhart, Ind., USA, and a 7 μm section isprepared in the cold.

Immunolabeling is performed with the following:

-   i. a first rabbit anti-human type I collagen antibody (dilution    1/40), and-   ii. a second anti-rabbit antibody coupled with FITC (Fluorescein    IsoThioCyanate) (dilution 1/160).-   DAPI (4′,6-diamidino-2-phenylindole dilactate) is used as a    counterstain.    3) Results

It is found that supports consisting of an aquatic matrix and a bovinematrix form more or less loose pores to which the fibroblasts adhere.

A greater proportion of fibroblasts is observed on the surface, forminga favorable covering over the dermis equivalent for the production ofreconstructed skin. The distribution of the fibroblasts is homogeneousin aquatic and bovine sponges.

In immunolabeling, it is found that the matrix formed of bovine collagenis labeled by the anti-human type I collagen antibody (crossing).

On the other hand, the matrix of aquatic origin is only very weaklylabeled by the anti-human collagen antibody.

The use of sponges composed of aquatic collagen therefore favorsidentification of the neosynthesized extracellular matrix.

These results are explained by Professor Hartmann's studies on thereactions of different antigens to different antibodies, determined bythe optical density measurements after immunolabeling which are givenbelow in Table IV, or Hartmann's table: TABLE IV Cross reaction withhuman, bovine and fish collagen (Elisa) Antigen Sole type I Human type IBovine I type Antibody collagen collagen collagen 20111 (225) 1/25 190 >815 1/50 210 > 548 1/100 73 1233 234 1/200 43 605 136 1/400 56 326 16550121 (03) 1/25 180 1550 > 1/50 130 1094 > 1/100 158 536 > 1/200 96 305967 1/400 109 215 728 50171 (01) 1/25 1880 64 73 1/50 1043 193 32 1/100571 51 33 1/200 523 51 87(>: optical density greater than 2000)

The results are expressed in OD×10³ (optical density at λ=450 nm).

Key: 20111 (225): anti-human type I collagen

50121 (03): anti-bovine type I collagen

50171 (01): anti-fish (sole) type I collagen

This Table of results shows that, irrespective of the antibody(anti-human type I collagen, anti-bovine type I collagen, anti-sole typeI collagen) in immunolabeling, the difference between human collagen andsole collagen is much greater than between human collagen and bovinecollagen. Consequently, in a fish collagen matrix, the collagensynthesized by human fibroblasts may be identified much more easily.This confirms the results described above which were obtained byimmunolabeling collagen synthesized in the fish collagen matrix with theanti-human type I collagen antibody, constituting a particularlyunexpected and advantageous result of the invention.

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 42. A supportfor tissue engineering comprising a porous matrix prepared from anaquatic collagen gel which has undergone a lyophilization step, andwherein the porous matrix is crosslinked by a chemical crosslinkingagent.
 43. The support of claim 42, wherein the crosslinking agent isselected from diphenylphosphorylazide or DPPA, a carbodiimide,N-hydroxysuccinimide, and glutaraldehyde.
 44. The support of claim 42,wherein the aquatic collagen is coming from teleost fish skin teleost.45. The support of claim 42, wherein the aquatic collagen is coming fromthe skin of a flat fish.
 46. The support of claim 42, wherein theaquatic collagen is coming from the skin of a flat fish industriallyfished selected from the sole, the dab, the turbot and the brill. 47.The support of claim 42, wherein the porous matrix comprises an aquaticcollagen admixed with chitosan or at least one glycosaminoglycan. 48.The support of claim 42, wherein said porous matrix is covered on atleast one side with an essentially compact collagen membrane selectedfrom a collagen film prepared by drying a second collagen gel in air orin gaseous fluid, and from a compressed collagen sponge.
 49. The supportof claim 42, wherein the porous matrix comprises living cells selectedfrom the group consisting of normal living cells, genetically modifiedliving cells and malignant living cells.
 50. The support of claim 49,wherein the living cells are selected from the group consisting offibroblasts, keratinocytes, melanocytes, Langerhans' cells originatingfrom the blood, endothelial cells originating from the blood, bloodcells, macrophages, lymphocytes, adipocytes, sebocytes, chondrocytes,osteocytes, osteoblasts and Merkel's cells originating from the blood.51. The support of claim 42, wherein the porous matrix contains livingfibroblasts selected from normal, genetically modified or malignantfibroblasts.
 52. The support of claim 48, wherein the essentiallycompact collagen membrane contains normal, genetically modified ormalignant living cells selected from keratinocytes, melanocytes,Merkel's cells originating from the blood, Langerhans' cells originatingfrom the blood, sebocyte cells originating from the blood, and nervecells.
 53. The support of claim 49, wherein the living cells originatefrom human subjects.
 54. The support of claim 51, wherein the livingcells originate from young subjects.
 55. The support of claim 51,wherein the living cells originate from old subjects.
 56. The support ofclaim 48, wherein the compression of the compressed collagen sponge iscarried out at a pressure of at least about 50 bar (50.10⁵ Pa) at atemperature of between 20° C. and 80° C.
 57. The support of claim 48,wherein the essentially compact membrane is prepared prior tocombination with the porous layer by preparing first the compactcollagen membrane and then by depositing it on said aquatic collagen gelbefore the combination of the collagen gel and of the compact collagenmembrane is frozen and lyophilized.
 58. The support of claim 42, in theform of a biomaterial, a reconstructing conjunctive tissue, or areconstructed skin.
 59. A method selected from a method for studyingcutaneous ageing, and a method for testing the efficacy of activesingredients with regard to slowing down or delaying cutaneous ageingcomprising the use of a support as defined in claim 42, wherein theporous matrix comprises living cells selected from the group consistingof normal living cells, genetically modified living cells and malignantliving cells.
 60. A method selected from a method for studying cutaneousageing, and a method for testing the efficacy of actives ingredientswith regard to slowing down or delaying cutaneous ageing comprising theuse of a support as defined in claim 48, wherein the porous matrixcomprises living cells selected from the group consisting of normalliving cells, genetically modified living cells and malignant livingcells.